Compositions comprising highly purified amphotericin B

ABSTRACT

A method for treating fungal infections in a mammal that includes administering a therapeutically effective amount of a composition comprising substantially pure amphotericin B and a pharmaceutically acceptable carrier. Amphotericin BHP is associated with decreased toxicity in mammals and cells measured by cell viability and expression of cytokine markers. Thus, this method allows for reduced adverse reactions when using amphotericin B products.

PRIORITY

This application claims priority to U.S. Patent Application No.60/415,671, filed on Oct. 3, 2002, the contents of which areincorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to the field of highly purifiedamphotericin B (amphotericin BHP) compounds, compositions, and methodsof use thereof. More specifically, the present invention relates to thetreatment of fungal infections in mammals.

The purification techniques of the present invention allow for theisolation of polyene anti-fungals from the supernatant of Streptomycesnodosus cultures.

BACKGROUND OF THE INVENTION

Amphotericin B products are used to treat a variety of fungalinfections, including systemic fungal infections.

However, amphotericin B induces serious adverse reactions. Thisbackground will describe the amphotericin B adverse reactions, discoveryof a mechanism for the adverse reactions, and a description of markersof these reactions.

Amphotericin B is used primarily an intravenous agent in the treatmentof severe fungal infections. However, its usefulness is compromised by ahigh incidence of adverse effects [flu-like symptoms (fever, chills,myalgias), capillary leak syndrome (hypotension, decreased organperfusion), pulmonary congestion, changes in mental status (lethargy,confusion, agitation), renal dysfunction with secondary hypokalemia,hypomagnesemia and anemia, and liver dysfunction]. These adversereactions are observed in up to seventy percent of treated patients. Themechanisms responsible for these reactions are, to date, not entirelyknown.

Through molecular biologic techniques, the present inventors havediscovered inflammatory cytokine genes that are up-regulated (increasedin cells) after exposure to amphotericin B. The genes includeinterleukin-1, a potent inflammatory cytokine. The adverse effectsassociated with stimulation of interleukin-1 are discussed below.

Proposed mechanisms of amphotericin B induced “flu like syndrome”include the expression of interleukin-1 (IL-1), tumor necrosis factor(TNF) or prostaglandins by mononuclear cells which then alter thehypothalamic set point inducing fever and chills. Administration ofendotoxin causes similar reactions. Amphotericin B-exposed mononuclearcells induces unique morphological changes and dramatically alteredprotein expression. Some host cell proteins have been reported to beinducible by amphotericin B, such as TNF-α and IL-1β. The presentinventors have demonstrated that this protein expression is notassociated with release of preformed protein; protein release isassociated with up-regulation of a gene or derepression of an inhibitorygene.

Currently, pharmacologic agents used to prevent amphotericin B adversereactions only address small aspects of the problem. Hydrocortisone isused to prevent the flu-like syndrome and hypotension. Acetaminophen isalso used to prevent the flu-like syndrome. Fluids administeredparenterally are used to prevent renal dysfunction. Also, lipid productshave been developed to decrease the toxicity of amphotericin B. Theseproducts encapsule or protect amphotericin B from causing thesereactions. However, they are not totally successful.

What is needed, then, is an amphotericin B product and treatment methodwithout the high incidence of side effects.

SUMMARY OF THE INVENTION

The invention is the discovery of an improved method of isolation foramphotericin B achieved through purification, called amphotericin BHP.Amphotericin BHP is associated with decreased toxicity in mammals andcells measured by cell viability and expression of cytokine markers.Thus, the present invention allows for reduced adverse reactions whenusing amphotericin B products. In certain embodiments of the presentinvention, amphotericin BHP is obtained from high pressure liquidchromatography fractionation.

One embodiment of the present invention is a pharmaceutical compositioncomprising substantially pure amphotericin B. For the purposes of thisinvention, substantially pure is greater than about 90% pure.Preferably, substantially pure amphotericin is greater than about 96%pure. In other embodiments, the amphotericin B is greater than about 97,98, or 99% pure.

Another embodiment of the present invention is a method of treatingfungal infections in a mammal, comprising administering atherapeutically effective amount of the highly purified amphotericin Bcompound of the present invention and a pharmaceutically acceptablecarrier to a mammal in need thereof.

Another embodiment of the present invention is a method of testingtoxicity in patients treated with pharmacological agents. Thisembodiment includes a method of testing patients treated withamphotericin B.

Another embodiment of the present invention is a pharmaceuticalcomposition comprising an amphotericin B formulation, wherein theamphotericin B formulation comprises no greater than about 4% by weightof impurities.

These and other embodiments will become clear from a reading of thedisclosure and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph that shows HPLC fractions.

FIG. 2 is a graph that shows viability of amphotericin B compositions ofthe present invention versus commercial formulations.

FIG. 3 is a graph that shows cytokine response induced by amphotericin Bcompositions of the present invention versus commercial formulations.

DETAILED DESCRIPTION OF THE INVENTION

As indicated above, while amphotericin B has been recognized as avaluable material, particularly in its powerful antifungal properties,its clinical (i.e. therapeutic use) has been limited because of thesevere side effects to the subject being treated. Amphotericin B isinsoluble in aqueous solution, consequently it is supplied commerciallyas a combination of Amphotericin B, lipid carriers, desoxycholate and/orbuffers, suspended in a glucose solution to form a colloidal suspensionfor administration to the patient. It is usually given intravenouslyover a period of from two to six hours. Faster infusions may result incardiotoxicity. Other toxic effects of Amphotericin B may manifestthemselves as renal disfunction, anemia, fever and hypotension.

The toxicity of Amphotericin B limits the total amount of the drug whichmay be used in the treatment of a fungal infection. Furthermore, it isoften ineffective in neutropenic and immunodeficient patients, patientswho are highly susceptible to fungal infections. Consequently, there isa need for a system which decreases the toxicity of Amphotericin B tothe mammalian system while simultaneously enhancing its effectivenessagainst the fungal infection.

It has recently been shown that the encapsulation of certain drugs inlipid carriers before administration to the patient can markedly alterthe pharmacokinetics, tissue distribution, metabolism and therapeuticefficacy of these compounds. Further, the distribution andpharmacokinetics of these drugs can be modified by altering the lipidcomposition, size, charge and membrane fluidity of the lipid carriers inwhich they are encapsulated.

The highly purified amphotericin B composition or complex of the presentinvention may be formed into pharmaceutical compositions, and may beadministered in accordance with known procedures of administeringamphotericin B products to a subject.

For example, the highly purified amphotericin B composition if thepresent invention may be formed into a lipid formation. Conventionalamphotericin B may be complexed with a lipid component that enablessolubility in aqueous solutions, thus allowing for parenteraladministration. Over the past two decades, researchers have investigatedthe utility of incorporating amphotericin B into phospholipid vesicles(liposomes) and/or cholesterol esters in order to provide larger amountsof parent drug and concomitantly, less nephrotoxicity. To date, at leastthree lipid formulations of amphotericin B are commercially available:amphotericin B lipid complex (ABLC, Abelcet); amphotericin B cholesterylsulfate complex, also called amphotericin B colloidal dispersion (ABCD,Amphotec); and liposomal amphotericin B (L-AmB, AmBisome). The presentinvention may be used in connection with each commercially availableformulation, and one of ordinary skill in the art would understand howto substitute the highly purified amphotericin B of the presentinvention for the commercially available amphotericin used in thecommercial formulation.

The current United States Food and Drug Administration (FDA) approveddosages for the three lipid formulations are believed to be as follows:L-AmB, 3-5 mg/kg/day; ABLC, 5 mg/kg/day; and ABCD, 3-4 mg/kg/day. Thelipid formulations can safely be administered at daily dosages 5 to 10fold higher than the daily dosages of amphotericin B.

The following chart identifies commercially available amphotericin Bdrugs that may incorporate the highly purified amphotericin product ofthe present invention:

TABLE 1 Examples of Commercially Available Amphotericin B DrugsManufacturer/ Generic Name Trade Name Marketer FDA Amphotericin BAmphotericin B (generic) 1958 deoxycholate (AmBD) Amphotericin B lipidABELCET ™ The Liposome 1995 complex (ABLC) Company Amphotericin BAMPHOTEC ™ SEQUIS 1996 cholesteryl sulfate Pharmaceuticals complex,amphotericin B colliodal dispersion (ABCD) Liposomal amphotericinAMBISOME ™ Fujisawa USA and 1997 B (L-AmB) NeXstar Pharmaceuticals

The chemical properties and physical characteristics of availableamphotericin B products are outlined in the Table 2, below:

TABLE 2 Chemical and Physical Properties of Examples of Amphotericin BDrugs Amphotericin Lipid Size B content Configuration (Nanometers) LipidComponent (mol %) AmBD Micelle <25 soduim deoxycholate — ABLCRibbon-Like 500–5000 dimyristoylphosphatidylcholine about 33%dimyristoylphosphatidylglycerol ABCD Disc-Like 125 cholesteryl sulfateabout 50% L-AmB Unilamellar  90 hydrogenated about 10% vesiclephosphatidylcholine (spherical) cholesteroldistearoylphospohatidlglycerol

The amphotericin BHP compositions of the present invention may beadministered to a patient in an amount ranging from about 0.001milligrams per kilogram of body weight per day to about 1000 mg per kgper day, including all intermediate dosages therebetween. It will bereadily understood that “intermediate dosages”, in these contexts, meansany dosages between the quoted ranges, such as about 0.001, 0.002,0.003, etc.; 0.01, 0.02, 0.03, etc.; 0.1. 0.2, 0.3, 0.4, 0.5, 0.6, 0.7,0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1,2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, etc.; 3, 4, 5, 6, 7, 8, 9, 10,etc.; 12, 13, 14, etc.; 50, 51, 52, 53, 54, etc.; 100, 101, 102, 103,104, etc.; 500, 501, 502, 503, etc.; 600, 700, 800, 900, and about 1000mg per kg per day, and including all fractional dosages therebetween.

In other embodiments, the amphotericin BHP compositions of the presentinvention may be administered to a patient in an amount ranging fromabout 0.01 milligrams per kilogram of body weight per day to about 100mg per kg per day, including all intermediate dosages therebetween.

In yet other embodiments of the present invention, the amphotericin BHPcompositions of the present invention may be administered to a patientin an amount ranging from about 0.1 milligrams per kilogram of bodyweight per day to about 10 mg per kg per day, including all intermediatedosages therebetween.

The pharmaceutical compositions of the present invention may beadministered by any known route, including parenterally and otherwise.This includes oral, nasal (via nasal spray or nasal inhaler), buccal,rectal, vaginal or topical administration. Administration may also be byorthotopic, intradermal subcutaneous, intramuscular, intraperitoneal orintravenous injection and/or infusion. Such compositions may beadministered as pharmaceutically acceptable compositions that includepharmacologically acceptable carriers, buffers or other excipients. Thephrase “pharmacologically acceptable” refers to molecular entities andcompositions that do not produce an adverse, allergic or other untowardreaction when administered to a human. For treatment of conditions ofthe lungs, the preferred route is aerosol delivery to the lung viabronchoalveolar lavage or the like.

Of course, with respect to conventional amphotericin intravenousinjection and/or infusion appears to be the most popular delivery route.In such embodiments, the amphotericin BHP compositions of the presentinvention may be administered gradually over a period of time rangingfrom 0.001 h to 100 h. In other embodiments, when administration of thepharmaceutical compositions of the present invention via intravenousinjection and/or infusion is the preferred route, the pharmaceuticalcompositions of the present invention should administered gradually overa period of time ranging from 0.1 h to 50 h. In other embodiments, whenadministration of the pharmaceutical compositions of the presentinvention via intravenous injection and/or infusion is the preferredroute, the pharmaceutical compositions of the present invention shouldadministered gradually over a period of time ranging from 1 h to 10 h.

As stated above, the highly purified amphotericin B of the presentinvention may be part of an HDLC (high drug:lipid ratio complexes). Asone example of this embodiment, the amphotericin BHP composition of thepresent invention may be used in the same manner as the amphotericin Bcomplex disclosed in in U.S. Pat. No. 6,406,713, incorporated herein byreference. Those embodiments include HDLC systems which comprise lipidsand bioactive agents including drugs. Such HDLCs may comprisephospholipids such as DMPC and DMPG, preferably in a 7:3 mole ratio orsaturated phospholipids or fatty acid phospholipids. The bioactive agentfor these embodiments is the highly purified amphotericin B of thepresent invention. Examples of the mole percent of the highly purifiedamphotericin B includes examples where the amount is from about 6 toabout 70 mole percent. Other example are in the about 30 to about 50mole percent. Pharmaceutical compositions of the HDLCs of the presentinvention may be made comprising pharmaceutical acceptable carriers ordiluents, and these compositions may be administered parenterally. Ofcourse, such compositions are used to treat infectious diseases such asfungal infections, by administering them to mammals such as humans. TheHDLC-containing compositions of the present invention include thosecompositions substantially free of liposomes and compositionssubstantially free of liposomes entrapping the drug. The term“substantially free” in this context shall be taken to mean generally nomore than about 10 percent by weight of liposomes, no more than about5%, and/or no more than about 3%.

Various methods for preparing the HDLCs of these embodiments aredisclosed in the U.S. '713 patent, including, for example, techniquesthat first solubilize the highly purified amphotericin B in a solventsuch as DMSO or methanol.

In an alternative method, lipid particles (or liposomes) containinghighly purified amphotericin B containing about 6 percent to 50 molepercent amphotericin B are formed and then the particles (or liposomes)are subjected to a heating cycle, at about 25° C. to about 60° C. Such acycle forms a more highly ordered and less toxic amphotericin B/lipidcomplex.

As further examples, the amphotericin BHP of the present invention maybe used in the manner and amounts described in U.S. Pat. Nos. 3,965,090;4,663,167; 4,766,046; 4,054,734; 5,965,156; 4,049,898; 5,194,266; and4,035,568, all of which are incorporated herein by reference.

The mode of administration of a preparation of the present invention maydetermine the sites and cells in the organism to which the amphotericinBHP compound will be delivered. Generally speaking, the highly purifiedamphotericin composition of the present invention will be administeredin admixture with a pharmaceutical carrier selected with regard to theintended route of administration and standard pharmaceutical practice.For instance, delivery to a specific site may be most easilyaccomplished by topical application (if the infection is external, e.g.,on areas such as eyes, skin, in ears, or on afflictions such as woundsor burns). Such topical applications may be in the form of creams,ointments, gels, emulsions, or pastes, for direct application to theafflicted area. Alternatively, the preparations may be injectedparenterally, for example, intravenously, intramuscularly, orsubcutaneously. For parenteral administration, they can be used, forexample, in the form of a sterile aqueous solution which may containother solutes, for example, enough salts or glucose to make the solutionisotonic. Other uses, depending on the particular properties of thepreparation, may be envisioned by those skilled in the art.

For therapeutic administration to humans, the prescribing physician willultimately determine the appropriate dosage for a given human subject,and this can be expected to vary according to the age, weight, andresponse of the individual as well as the nature and severity of thepatient's symptoms. in the curative or prophylactic treatment of fungalor viral diseases, The dosage of the drug in the HDLC or liposomal formwill generally be about that employed for the free drug. In some cases,however, it may be necessary to administer dosages outside these limits.The prescribed amount may vary when used for curative and prophylactictreatment of fungal or other infectious diseases.

The following examples are submitted to show embodiments of the presentinvention. They are intended to show embodiments of the presentinvention and be exemplary of aspects of the present invention and notintended to be limiting thereof.

The examples illustrate other embodiments of the present invention andare designed as an in vitro evaluation of amphotericin B componentsfractionable through high pressure liquid chromatography using thedescribed methods.

EXAMPLE 1 Reagents

Aliquots of about 11.2 mg of powder are measured from commercial gradeamphotericin B manufacturers. In this example, Amphotericin Bmanufactured by Apothecon and Sigma are utilized. These aliquots containabout 5 mg of amphotericin B. Each sample is stored at about 4° C. inmicrocentrifuge tubes and diluted immediately prior to each experiment.The 5 ug/mL dilution is made by adding about 1.0 mL sterile water toeach aliquot. The 2.5 ug/mL is made by using about a 2:1 dilution ofabout 500 uL of the 5 ug/mL stock. The Sigma brand is not apharmaceutical grade product and is selected as a positive controlrepresenting a “minimally purified” product. Fresh aliquots are dilutedprior to each experiment and agitated immediately before use. A samplealiquot from each amphotericin B preparation is reserved for testingendotoxin contamination.

Other pharmacologic agents utilized include Escherichia coli endotoxin(Serotype 026:b6 lipopolysaccharide, Sigma; St. Louis, Mo.) (LPS),desoxycholate and sodium phosphate buffer which are obtained from Sigma.These reactions are used as positive and negative controls for IL-1βexpression from mononuclear cells. Reagents used in the IL-11βexpressionassay are diluted with sterile water so that 0.01 ml of stock solutionsadded to culture wells resulted in the final concentrations noted.

EXAMPLE 2 Mononuclear Cell Preparation

Unsupplemented media (RPMI-1640) is obtained from Flow Laboratories(McLean, Va.). Mononuclear cells [THP-1; ATCC 222:U937] is resuspendedto a final concentration of approximately 5×10⁶ cells/mL in supplementedmedia (RPMI-1640, about 10% autologous serum, about 100 ug/mLstreptomycin and about 100 u/mL penicillin). About one milliliter ofmononuclear cells is seeded in Limbro 24-well plates (Flow Laboratories;McLean, Va.) and incubated for 24 hours at 37° C. in 5% CO₂.

EXAMPLE 3 Assays

High Pressure Liquid Chromatography Isolation and Validation:Amphotericin B aliqotes of Example 1 are applied to a 4.6×150 mm 5micron AquaC18™ column (Phenomenex®), and components resolvedisocratically using about 70% methanol:about 30% 5 mM sodium citrate (pH7.0) (vol:vol) flowing at a rate of about 1 ml per minute. Column eluantis monitored at about 305 nm and about 405 nm. Specific composition ofthe solvent varies from about 70:30 (methanol:sodium citrate) to about75:25 (methanol:sodium citrate) to achieve the best resolution of highpurity amphotericin B and other components.

Viability Assay: Amphotericin B fractions are tested for effects ofhuman cell viability. A tritriated thymidine incorporation assay inaddtion to Erythrocin red or Trypan Blue are exclusion dye tests isused. Assays of cytotoxic activity using tritiated thymidine areperformed under aerobic conditions. Substances to be assayed are dilutedin RPMI containing about 10% human or bovine serum, added to the firstvertical column of a 96-well flat-bottom tissue culture plate, andserially diluted in RPMI in the remaining wells of each row using amulti-tip pipetter. Control rows without drugs or containingsolubilizing agents (DMSO, DOC, glycerin) are similarly treated so thatthe concentrations of these diluents are similarly decreased across theplate. Logarithmic growth phase THP-1 cells are added (about 10⁶ cellper well), and the plates maintained in a CO₂ incubator (about 5% CO₂ inair; about 37° C.) for about 24 hours. At the end of this drug-exposurephase, the plates are centrifuged about 5 min at about 200×g, thesupernatants are briskly decanted, and the wells refilled with PBS.After the third such rinse, the wells are refilled with drug-free RPMI.The plates are returned to the CO₂ incubator, and any remaining viablecells allowed to grow during a 24-hour amplification phase. The platesare then centrifugally washed twice with PBS, and the wells refilledwith RPMI-1640 (Sigma) containing about 10% FBS and about 5 mCi/well[methyl-³H] thymidine (New England Nuclear, Boston, Mass.) and incubatedfor about an additional hour. The plates are then harvested toglass-fiber paper using distilled water rinses, and the paper countedwith a Matrix₉₆ gas-ionization direct-beta counter (Packard InstrumentCo, Meriden Conn.). Each drug exposure is done in triplicate rows of asingle plate. Each plate included controls for the cytotoxicity ofsolubilizing agents (DMSO, DOC, glycerin) and a drug-free control.Radioactivity per well is analyzed with Microsoft-Excel. The assaydifferentiates target cell death from drug-induced loss of motilitysince it measured incorporation of radioactivity into newly synthesizedDNA after drug washout. Minimum lethal concentration (MLC) is defined asthe lowest concentration of drug (in a 1:2 serial dilution series) tokill all the target cells (reduce incorporation to background levels).The 100% control level (no killing) for each plate is defined as themean radioactivity per well of the drug-free control row. Experimentalresults are expressed as percent of the control mean using the drug-freecontrol as 100%. This assay is adapted for use with mammalian cells intissue culture monolayers by substituting DME containing 10% FBS for theRPMI and by extending the tritiated thymidine pulse labeling period inRPMI/10% FBS to an overnight incubation.

In vitro Infusion Related Reaction Assay: Amphotericin B is added toLimbro 24-well plates at final concentrations of about 0 ug/mL and 20ug/mL. Cells are then incubated for about 2 hours. Supernatants arecollected from each well after three freeze-thaws and stored at about−70° C. until assay. Samples are assayed for IL-1β using anenzyme-linked immunosorbent assay (Cistron Biotechnology; Pine Brook,N.J.) (ELISA). The procedure involves a four-step test carried out inmicrotiter wells which are coated with IL-1β specific monoclonalantibody. Manufacturer's data indicate an assay sensitivity of 20.0pg/mL and a specificity for IL-1β. There is no cross-reactivity forIL-1α, IL-2, TNF-α or interferon. Evaluations of assay precisiondemonstrate a coefficient of variation of about 5.3% to about 6.7% forintra-assay variability and about 6.6% to about 8.4% inter-assayvariability. Data are the mean of duplicate assays and are expressed inpg/mL on the basis of standards supplied by the manufacturer.

Amphotericin B Assay: A polyclonal rabbit antibody is isolated from NewZealand white rabbits after standard immunization with amphotericin Bcomplexed with an immune adjuvant, Keyhole Limpet Hemocyanin. Ananti-amphotericin B antibody is purified by filtering serum through anAminolink Affinity-Pak Column (ImmunoPure Ag/Ab; Pierce Chemical Co.,Rockford, Ill.), diluted to a final concentration of 180 ug/mL andfrozen at about −70° C. until further use. The ELISA is initiated byremoving a light protection cover from an amphotericin B-bovine serumalbumin coated microtiter plate. The 1.0 ug/mL coating solution isemptied, and triplicate well washings are performed. Wash solutionconsisted of a standard phosphate buffered salt solution containingtween. The 96-well plates are then blocked with bovine serum albumin forone hour at about 37° C. and again thrice washed. Addition ofamphotericin solution (100 ul) is followed by Anti-amphotericin Bantibody (100 ul) and then incubation for about an hour at about 37° C.Plates are emptied and thrice washed. Horseradish peroxidase-anti-rabbitIgG diluted 1:1000 in buffer is added after blot drying. Plates areagain incubated for an additional hour at about 37° C. and thricewashed. A 200 uL aliquot of peroxidase substrate solution [Fast-P-9187;Sigma Chemical, St. Louis, Mo.] is pipetted into each well and ELISAplates are tested after about 10 minutes for photometric density[Dynatech multiscan; Flow Laboratories, McLean, Va.] utilizing a 405 nmfilter. Finally, amphotericin B-spiked samples are tested for stabilityduring storage at about −70° C. for about 60 days. Triplicatelight-protected samples at amphotericin B concentrations of 2.5 ug/mLand 5 ug/mL are assayed by ELISA.

Published data indicate an assay sensitivity of 0.15 ug/mL. There iscross-reactivity for agents with polyene structures, nystatin andhamycin. Evaluations of assay precision demonstrates a coefficient ofvariation of 3.0% for intra-assay variability. Data are the mean ofduplicate assays and are expressed in ug/mL on the basis of Apotheconbrand as the standard.

Spectrophotometric Assay: Classification and quantification of a polyenecan be performed based on each agent's ultraviolet absorption. Theultraviolet spectra for tetraenes (nystatin, amphotericin A) hascharacteristic peaks at about 290 nm, about 305 mu and about 318 mu,while heptaenes (amphotericin B) occurs at about 360 nm, about 378 nmand about 405 nm. The principles of Beer's Law are used to estimate therelative amount of amphotericin A or B in each preparation. Nystatin isused in our assays for amphotericin A and tested at an optical densityof about 290 nm. Amphotericin B formulated by Apothecon is tested at anoptical density of about 360 nm. These optical densities are selectedowing to their uniqueness to amphotericin A and B, respectively. Peaksat other optical densities are shared with pentaenes and hexaenes,making it difficult to preclude their existence in the solution. Twounique peaks for pentaenes are identified at optical densities of about325 nm and about 333 nm. Samples of each amphotericin formulation arediluted in about 1.0 mL DMSO, then further diluted with about 5 mLmethanol. A 400 uL aliquot from this dilution is further diluted inabout 5 mL of methanol. A dilution of sodium desoxycholate does notaffect the optical density measurements. Ultraviolet light absorbancefrom about 200 nm to about 450 nm is determined with a GilfordSpectrophotometer and analyzed by Response II software (Gilford; Dayton,Ohio). Data are the mean of triplicate assays, and amphotericinconcentrations are expressed in micrograms per milliliter on the basisof Apothecon brand as the standard for amphotericin B and nystatin asthe standard for amphotericin A.

EXAMPLE 4 Susceptibility Testing

Antifungal Agents: Stock solutions of sample agents are prepared bydissolving stock powder in dimethyl sulfoxide (DSMO) and appropriatelydiluted using RPMI 1640 buffered to about pH 7.0 with about 0.165Mmorpholinepropanesulfonic acid (MOPS) (PML Microbiologicals,Wilsonville, Oreg.). The final concentration of DMSO is such that theconcentration in test solutions comprised less than about 6% of thetotal solution composition.

Test Isolates: One Candida albicans isolate, American Type CultureCollection (ATCC) strain 90028, is selected.

DMSO inhibitory test: Effects of DMSO on MIC determination are testedusing NCCLS broth microdilution guidelines and the test isolate C.albicans 90028. One hundred microliters of a DMSO and an RPMI solutionare placed in the wells of a microdilution tray so that theconcentrations of DMSO in tested wells are 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 15, 20, 25, and 30 percent. Yeast inoculum is prepared as describedin antifungal susceptibility testing, and 100 gl are added to each testwell. The trays are incubated in a humid, dark chamber for about 48hours at about 35° C. The MIC is determined as any visible change ingrowth when compared to control. DMSO inhibitory tests are preformed induplicate.

Antifungal Susceptibility Testing: Sample MIC's are determined by brothmicrodilution according to NCCLS guidelines. Isolates are subculturedtwice on potato dextrose agar (PDA) plates (Remei, Lenexa, Kans., USA).Fungal suspensions are prepared by transferring four to five coloniesinto about 5 mL of sterile about 0.9% saline. The suspensions arestandardized, using spectrophotometric methods, and diluted in RPMI 1640buffered to about pH 7.0 with 0.165M MOPS (PML Microbiologicals,Wilsonville, Oreg.) to yield an initial inoculum of 0.5×10³-2.5×10³CFU/mL. 100 uL of inoculum is added to each well of a microtitre traycontaining 100 uL of serially diluted drug in RPMI solution. Sampleconcentrations of about 0.0039, about 0.0078, about 0.0156, about0.0312, about 0.0624, about 0.125, about 0.25, about 0.5, about 1, about2, and about 4 ug/mL are achieved. The trays are incubated in a humid,dark chamber for about 48 hours at about 35° C. The MICs sre recorded asthe wells with 80% inhibition and 100% inhibition when compared tocontrol. MIC determinations are performed a minimum of two times with anadditional run if the quantity of sample drug allowed for it. It isdecided to determine the MFC on samples which achieved definitive MICs.After the MIC is read at 48-h, a 1 uL sample is withdrawn from eachmicrotiter tray with a 96 pin replicator (Boekel, Feasterville, Pa.) andplated onto an RPMI agar plate. Samples are incubated in a humid, darkchamber for about 48 hours at about 35° C. MFC is determined by thecomplete lack of organism growth on the plate and recorded as thecorresponding well in the MIC tray.

EXAMPLE 5 Statistical Analysis

An ANOVA is performed comparing Apothecon brand amphotericin Bformulation with Sigma brand amphotericin B. A standard T-test is usedto compare the differences identified within the ANOVA. An alpha ofabout 0.05 and a beta of about 0.2 are selected for this comparison.Sigma Stat [Jandel; San Dimas, Calif.] is utilized for the statisticalanalysis.

EXAMPLE 6 Results

High pressure liquid chromatography of amphotericin B commericallymanufactured leads to the identification of multiple amphotericinproducts and probably bacterial/fungal endotoxin (Figure I). Isolationof each peak at optical densities of about 305 nm and about 405 nm hasled to the identification of these products as polyene compounds orendotoxin. The fractions are isolated from aliquotes from commercialbrand amphotericin B or Sigma brand amphotericin B resolves with usingabout 70% methanol:30% 5 mM sodium citrate (pH 7.0) (vol:vol) at a flowrate of about 1 mL/minute. The fraction elutes from the column at about12 minutes from a 4.6×150 mm 5 micron AquaC18™ column monitored at about305 nM and about 405 nM. This component is highly purified amphotericinB (Amphotericin BHP).

Viability Assay

Viability of cells is directly related to the quantity of DMSO used tosolubilize amphotericin BHP. Therefore, only concentrations less thanabout 6% are utilized. In addition, deoxycholate solution is used tosolubilize amphotericin BHP. Doxycholate did not cause decreases inviability compared to control at amounts less than about 20.2 mg perabout 50 mg amphotericin B. The viability of THP-1 cells exposed toamphotericin BHP at concentrations ≦5 ug/in L is greater than theviability in cells exposed to the same contrations of commercial brandor Sigma brand amphotericin B (Figure II).

In vitro Infusion Related Reaction Assay

The potential of each of the amphotericin B formulations to induceinfusion-related reactions measured by IL-1β expression are displayed inFigure III. The amount of IL-1β expression associated with THP-1 cellsexposed to the individual amphotericin B formulations has beenstandardized to the Apothecon formulation and is represented as percentexpression. Control cells exposed to sterile water or lipid express nomore than about 120 pg/mL of IL-1β while Apothecon brand amphotericin Bcause the expression of about 300 pg/mL and 750 pg/mL IL-1β for the 0.1ug/mL and 10.0 ug/mL concentrations, respectively. Less cytokine isexpressed in response to amphotericin BHP compared to the otherformulations (Apothecon and Sigma) The difference is signficiantcompared to Apothecon brand amphotericin B.

Amphotericin B Assay

Quantification of amphotericin B in stock vials is completed withinabout 12 hours of the IL-1β. Apothecon brand amphotericin B ismeasurable within about 5% of the reported values by the manufacturer.One concentration (5 ug/mL) of Sigma brand is measured at about39.5±21.05 ug/mL. In addition, Sigma brand amphotericin B is determinedto be greater than about 5% of the manufacturers labeling in all samplestested. These data, suggest that there are several polyene antifungalsin Sigma brand amphotericin B which are identified by our Amphotericin BELISA and that have spectrophotometric patterns distinct fromamphotericin B.

Spectrophotometric Assay

Quantitification of amphotericin A, B, and BHP is completed byspectrophotometry. Nystatin is used as the tetraene control representingamphotericin A and Apothecon brand as the standard for the hepateneamphotericin B. The value obtained by utilizing Beer's Law (USPequation) to calculate amphotericin A should represent the amount ofamphotericin A relative to the Apothecon brand. No significantdifferences in A or B could be found in any of the amphotericin Bformulations utilized clinically (data not shown). Sigma brandamphotericin B did contain significantly more of the amphotericin Acompound in comparison to the commercial brands. Based on thesespectrophoto-metric results, one could hypothesize that the AmphotericinB ELISA actually effectively measures polyene antifungals as a class.However, the presence of amphotericin A is not the complete explanationfor the amphotericin measured in the ELISA. There are positivecorrelations between both amphotericin B concentration and theconcentrations of amphotericin A (r²=0.8831; p<0.01) or interleukin-1βconcentration (r²=0.9633; p<0.01). Amphotericin BHP demonstrated apattern consistent with polyene antifungals.

Spectrophotometric evaluations of the unique absorption maximum for eachof these polyenes, except for the hexaenes, are performed. The hexaenesshare all spectrophotometric peaks with either a pentaene or a heptaene.Using Beers' Law, we calculated the amount of each polyene relative toApothecon's product. Pentaene (unique O.D. peak=333) quantities arehighest in the Sigma brand (about 167%) relative to Apothecon. Heptaene(unique O.D. peak=405) content is negligible (about <6.7%) in all exceptthe Sigma Brand (about 10.8%). There are distinct peaks at opticaldensities of 345, 363 and 386 that correspond to either a pentaene or ahexaene. At these peaks (345, 363 and 386), differences in the productscould be easily visualized (Figure III). The peak obtained at about 12minutes is a polyene antifungal. The peaks that occur at other timepoints are also polyenes.

Bacterial endotoxin contamination could explain an increased observationof infusion related reactions when switching between products.Amphotericin B manufactured by Lyphomed Pharmaceuticals in the early1990s is identified as possibly contaminated. Therefore, we are diligentin testing our reagents for endotoxin contamination. Samples fromdiluent (sterile water), media, reagents and culture plates are assayedfor endotoxin by the manufacturer utilizing the limulus amebocytelysate. The limulus amebocyte lysate (LAL: Associates of Cape Cod Inc.;Woodshole, Mass.) testing employed an Escherichia coli endotoxinstandard with a lower limit of detection of 3.0 pg/ml. Amphotericin Binterferes with the LAL assay by disrupting the clot formation and clotadhesion, rendering this test difficult to perform and interpret. Atconcentrations achievable during clinical use, amphotericin Binterference appears to be minimal. Testing of the Sigma brand and theApothecon brand of amphotericin B preparations at 2.5 ug/mL and 5 ug/mLfound no endotoxin.

Susceptibility Testing

DMSO inhibitory test. The results of the DMSO inhibitory tests displayedcomplete inhibition of growth at about 8% DMSO and partial inhibition atabout 7%. The well with about 6% DMSO showed no visible difference fromcontrol. Therefore about 6% is set as the maximum allowable amount ofDMSO in any well. Whenever possible lower concentrations are used, butdue to the limited sample sizes, levels near about 6% are often used inthe well with the highest drug concentration.

Antifungal Susceptibility Testing. Antifungal agents are prepared justprior to testing in all cases except in that of sample #1. After theaddition of DMSO, it appeared that the drug had not completelysolubilized. It is allowed to sit in the dark at room temperature forabout 48 hours. The predetermined amount of RPMI is then added to thetube. An apparent exothermic reaction took place, releasing heat, andthe remaining pellet dissolved completely. The sample is then usednormally in susceptibility testing.

Other limitations are caused by the relatively small size of the sampledrugs. Samples 2-7 are performed in duplicate and samples 1,8-12 areperformed in triplicate. These low concentrations also made itimpossible to test the full range of drug concentrations for eachsample. Samples 3 and 6 are only tested as high as 1 ug/ml. and samples2,5, and 7 are tested up to about 2 ug/ml. Finally, as previouslydiscussed, the sample size required higher concentrations of DMSO thannormally utilized.

TABLE 3 Activity of HPLC fractions against Candida albicans ¹ ELUTIONTIME Median MIC₈₀ Median MIC₁₀₀ MFC^(1,2) 1 >4 >4 NT 2 2 >2 NT 3 >1 >1NT 4 >4 >4 NT 5 >2 >2 NT 6 >1 >1 NT 7 >2 >2 NT 8 1.00 2 2 9 1.00 2 1 10 0.25 2 0.5 11  0.25 0.5 0.5 12  0.5 2 2 Solvent NA NA % NA AmB 0.10 0.250.625 ¹tested against Candida albicans B311 ²tested against Candidaalbicans ATCC 90028 (NCCLS-recommended strain) MIC = Concentration(μg/ml) that inhibits 80% growth MFC = Concentration (μg/ml) that isfungicidal to all cells (no growth is seen on agar plates) NT = nottried due to inactivity NA = not active AmB = Amphotericin B High PurityTime = elapsed time in minutes. MIC DATA RESPRESENT THE AVERAGE OF THREETESTS

Median susceptibility results are shown in Table 3. Results with a “>”sign signify that no inhibition is observed at or below these testedlevels. Sample #2 displays a “2” for MIC₈₀ meaning that an 80% reductionis observed in this well. Though not indicated in the table, sample #2causes some reduction at 2 ug/mL.

It will be apparent to those skilled in the art that variousmodifications and variations can be made in the present inventionwithout departing from the scope or spirit of the invention. Otherembodiments of the invention will be apparent to those skilled in theart from consideration of the specification and practice of theinvention disclosed herein. It is intended that the Specification andExample be considered as exemplary only, and not intended to limit thescope and spirit of the invention.

Unless specifically otherwise indicated, all numbers expressingquantities of ingredients, properties such as reaction conditions, andso forth used in the Specification and Claims are to be understood asbeing modified in all instances by the term “about.” Accordingly, unlessindicated to the contrary, the numerical parameters set forth in theSpecification and Claims are approximations that may vary depending uponthe desired properties sought to be determined by the present invention.

Notwithstanding that the numerical ranges and parameters setting forththe broad scope of the invention are approximations, the numericalvalues set forth in the experimental or example sections are reported asprecisely as possible. Any numerical value, however, inherently containcertain errors necessarily resulting from the standard deviation foundin their respective testing measurements.

Throughout this application, various publications are referenced. Allsuch references are incorporated herein by reference.

1. A method for treating fungal infections in a mammal, comprising:providing a composition that comprises an active ingredient thatincludes Amphotericin B, wherein the Amphotericin B compound is presentin an amount greater than 96% with no greater than 4% of impurityproducts, and a pharmaceutically effective carrier; and administering atherapeutically effective amount of said composition to a subject inneed thereof.
 2. The method of claim 1, wherein the amphotericin B ispresent in an amount greater than about 98% and no greater than about 2%of impurity products.
 3. The method of claim 1, wherein saidpharmaceutically acceptable carrier is a lipid carrier.
 4. The method ofclaim 1, wherein the administering step is intravenous.
 5. Apharmaceutical composition, comprising: an active ingredient that has atleast 96% of an amphotericin B compound and no greater than 4% ofimpurity products, and a pharmaceutically acceptable carrier.
 6. Thecomposition of claim 5, wherein the amphotericin B compound is presentin an amount greater than about 98%.
 7. The method of claim 1, whereinthe impurities comprise at least one of a non-amphotericin B polyenecompound or an endotoxin compound.
 8. The composition of claim 5,wherein the impurities comprise at least one of a non-amphotericin Bpolyene compound or an endotoxin compound.
 9. A pharmaceuticalcomposition that comprises: an active ingredient that is at least 96%w/w of a compound of the following formula:

and a pharmaceutically acceptable carrier.
 10. The composition of claim9, wherein said pharmaceutically acceptable carrier is a lipid carrier.11. The composition of claim 9, the active ingredient comprising nogreater than 4% w/w of at least one of a non-amphotericin B polyenecompound or an endotoxin compound.
 12. A method for treating fungalinfections in a mammal, comprising: providing a composition thatcomprises an active ingredient that has at least 96% amphotericin Bpolyene compound and a pharmaceutically effective carrier; andadministering a therapeutically effective amount of said composition toa subject in need thereof.
 13. The method of claim 12, wherein theactive ingredient that is at least 96% w/w of a compound of thefollowing formula:

and a pharmaceutically acceptable carrier.
 14. The method of claim 12,wherein said pharmaceutically acceptable carrier is a lipid carrier. 15.A pharmaceutical composition that comprises: an active ingredient thatis at least 96% w/w of a compound of the following formula:

comprising no greater than 4% w/w of at least one of a non-amphotericinB polyene compound or an endotoxin compound, and a pharmaceuticallyacceptable carrier.
 16. The composition of claim 15, wherein saidpharmaceutically acceptable carrier is a lipid carrier.
 17. Thecomposition of claim 15, wherein the active ingredient is at least 98%w/w of a compound of the following formula:

and comprises no greater than 2% w/w of at least one of anon-amphotericin B polyene compound or an endotoxin compound; and and apharmaceutically acceptable carrier.